Allosteric hemoglobin modifiers which decrease oxygen affinity in blood

ABSTRACT

A family of compounds has been found to be useful for right-shifting hemoglobin towards a low oxygen affinity state. The compounds are capable of acting on hemoglobin in whole blood. In addition, the compounds can maintain the oxygen affinity in blood during storage and can restore the oxygen affinity of outdated blood.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation application of the co-pendingU.S. patent application entitled "Using Allosteric Hemoglobin ModifiersTo Decrease Oxygen Affinity in Blood" having U.S. Ser. No. 07/885,721,which was filed May 18, 1992, and which issued as U.S. Pat. No.5,248,785. That patent application is itself a continuation-in-part(CIP) of Ser. No. 702,947, filed May 20, 1991 now U.S. Pat. No.5,122,519 patent application is itself a continuation-in-partapplication of Ser. No. 478,848 filed Feb. 12, 1990 now U.S. Pat. No.5,049,695. The subject matter of this application is also acontinuation-in-part of the co-pending U.S. patent application entitled"ALLOSTERIC HEMOGLOBIN MODIFIER COMPOUNDS" having Ser. No. 07/722,382which was filed Jun. 26, 1991, and which itself is a continuation of theU.S. patent application having Ser. No. 07/623,346 which was filed Dec.7, 1990. The text of all three of the above-identified patentapplications and U.S. Patent is herein incorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention generally relates to using a family of compoundsto adjust the allosteric equilibrium of hemoglobin toward a low oxygenaffinity state. Moreover, the invention contemplates using the family ofcompounds for use in treating diseases involving oxygen deficiency, inwound healing, and in restoring oxygen affinity of stored blood.

2. Description of the Prior Art

Hemoglobin is a tetrameric protein which delivers oxygen via anallosteric mechanism. Oxygen binds to the four hemes of the hemoglobinmolecule Each heme contains porphyrin and iron in the ferrous state. Theferrous iron-oxygen bond is readily reversible. Binding of the firstoxygen to a heme requires much greater energy than the second oxygenmolecule, binding the third oxygen requires even less energy, and thefourth oxygen requires the lowest energy for binding. Hemoglobin has twoα and two β subunits arranged with a two fold symmetry. The α and βdimers rotate during oxygen release to open a large central watercavity. The allosteric transition that involves the movement of thealpha-beta dimer takes place between the binding of the third and fourthoxygen. The α₁ β₁ interface binding is tighter than the α₁ α₂ or α₁ β₂interfaces.

In blood, hemoglobin is in equilibrium between two allostericstructures. In the "T" (for tense) state, hemoglobin is deoxygenated. Inthe "R" (for relaxed) state, hemoglobin is oxygenated. An oxygenequilibrium curve can be scanned, using well known equipment such as theAMINCO™ HEM-O-SCAN, to observe the affinity and degree of cooperativity(allosteric action) of hemoglobin. In the scan, the Y-axis plots thepercent of hemoglobin oxygenation and the X-axis plots the partialpressure of oxygen in millimeters of mercury (mm Hg). If a horizontalline is drawn from the 50% oxygen saturation point to the scanned curveand a vertical line is drawn from the intersection point of thehorizontal line with the curve to the partial pressure X-axis, a valuecommonly known as the P₅₀ is determined (i.e., this is the pressure inmm Hg when the scanned hemoglobin sample is 50% saturated with oxygen).Under physiological conditions (i.e., 37° C., pH=7.4, and partial carbondioxide pressure of 40 mm Hg), the P₅₀ value for normal adult hemoglobin(HbA) is around 26.5 mm Hg. If a lower than normal P₅₀ value is obtainedfor the hemoglobin under test, the scanned curve is considered to be"left-shifted" and the presence of high affinity hemoglobin isindicated. Conversely, if a higher than normal P₅₀ value is obtained forthe hemoglobin under test, the scanned curve is considered to be"right-shifted" and the presence of low affinity hemoglobin isindicated.

It has been proposed that influencing the allosteric equilibrium ofhemoglobin is a viable avenue of attack for treating diseases. Theconversion of hemoglobin to a high affinity state is generally regardedto be beneficial in resolving problems with deoxy Hemoglobin-S (sicklecell anemia). The conversion of hemoglobin to a low affinity state isbelieved to have general utility in a variety of disease states wheretissues suffer from low oxygen tension, such as ischemia and radiosensitization of tumors. Several synthetic compounds have beenidentified which have utility in the allosteric regulation of hemoglobinand other proteins. For example, several new compounds and methods fortreating sickle cell anemia which involve the allosteric regulation ofhemoglobin are reported in U.S. Pat. No. 4,699,926 to Abraham et al.,U.S. Pat. No. 4,731,381 to Abraham et al., U.S. Pat. No. 4,731,473 toAbraham et al., U.S. Pat. No. 4,751,244 to Abraham et al., and U.S. Pat.No. 4,887,995 to Abraham et al. Furthermore, in both Perutz, "Mechanismsof Cooperativity and Allosteric Regulation in Proteins", QuarterlyReviews of Biophysics 22, 2 (1989), pp. 163-164, and Lalezari et al.,"LR16, a compound with potent effects on the oxygen affinity ofhemoglobin, on blood cholesterol, and on low density lipoprotein", Proc.Natl. Acad. Sci., USA 85 (1988), pp. 6117-6121, compounds which areeffective allosteric hemoglobin modifiers are discussed. In addition,Perutz et al. has shown that a known antihyperlipoproteinemia drug,bezafibrate, is capable of lowering the affinity of hemoglobin foroxygen (see, "Bezafibrate lowers oxygen affinity of hemoglobin", Lancet1983, 881.

German Patent Applications 2,149,070 and 2,432,560, both to Witte etal., disclose compounds which are structurally similar to some of thecompounds in the family of compounds defined by this invention. However,the Witte et al. patent applications contemplate use of the compoundsfor the reduction of serum lipid levels. The Witte et al. patentapplications do not provide any indication of the potential use of thecompounds for allosteric hemoglobin modification.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide a methodof using a family of compounds to allosterically modify hemoglobin suchthat the hemoglobin is present in blood in a lower oxygen affinitystate.

It is another object of the present invention to provide a method ofprolonging the storage life of blood by adding compounds within aparticular family of compounds to the blood.

According to the invention, an allosteric hemoglobin modifying family ofcompounds is defined by the formula: ##STR1## where R₂ is a substitutedor unsubstituted aromatic such as a phenyl, naphthyl, or indanyl, or aheterocyclic aromatic, or a substituted or unsubstituted alkyl ringcompound such as a cyclohexyl or adamantyl, or a substituted orunsubstituted phthalimide compound where X is a carboxyl, Y is anitrogen and R₂ completes the phthalimide compound by being bonded toboth X and Y, and where X, Y, and Z are CH₂, NH, CO, O or N with thecaveat that the X, Y, and Z moieties are each different from oneanother, and where R₁ has the formula: ##STR2## where R1 can beconnected to any position on the phenyl ring and R₃ and R₄ are hydrogen,halogen, methyl, or ethyl groups and these moieties may be the same ordifferent, or alkyl moieties as part of an aliphatic ring connecting R₃and R₄, and R₅ is a hydrogen, loweralkyl such as methyl, ethyl orpropyl, or a salt cation such as sodium, potassium, or ammonium. Manycompounds within this family have been synthesized and their effect onthe P₅₀ value of hemoglobin has been determined. Each of the compoundstested was found to increase the P₅₀ value of hemoglobin; hence, thecompounds are capable of driving the allosteric equilibrium ofhemoglobin towards a condition favoring the low oxygen affinity state.In addition, the compounds were found to stabilize the degree of oxygendissociation of hemoglobin in stored blood over extended periods oftime. Furthermore, the compounds were found to be well tolerated by micewhen administered as an intraperitoneal dose. Because the compoundswithin the family defined by this invention are capable of shifting thehemoglobin allosteric equilibrium toward the low affinity "T" state,they have the ability to cause hemoglobin to deliver more oxygen totissues. Thus, the compounds of the invention should be valuable asantiischemic agents, as sensitizers for x-ray irradiation in cancertherapy, as wound healing agents, in treating disorders related to lowoxygen delivery in the brain such as Alzheimer's, depression, andschizophrenia, in preparing blood substitutes, and in blood storage.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, aspects and advantages will be betterunderstood from the following detailed description of a preferredembodiment of the invention with reference to the drawings, in which:

FIG. 1a is a chemical structure defining a particularly preferred groupwithin the family of compounds used in the present invention;

FIGS. 1b and 1c are chemical structures defining two subsets of thefamily defined in FIG. 1a;

FIGS. 2a-b depict chemical structures of precursor compounds arranged inreaction schemes for preparing compounds that are useful asintermediates for synthesizing compounds within a first group of thefamily of compounds;

FIG. 2c depicts chemical structures, including the intermediatesproduced as shown in FIGS. 2a-b, arranged in a reaction scheme toprepare the first group of preferred compounds;

FIG. 3 depicts chemical structures arranged in a reaction scheme toproduce a second group of the family of preferred compounds;

FIG. 4 depicts chemical structures arranged in a reaction scheme toproduce a third group of the family of preferred compounds;

FIGS. 5a-b depict chemical structures of precursor compounds arranged inreaction schemes for preparing compounds that are useful asintermediates for synthesizing compounds within a fourth group of thefamily of preferred compounds;

FIG. 5c depicts chemical structures, including the intermediatesproduced in FIGS. 5a-b, arranged in a reaction scheme to produce thefourth group of compounds;

FIG. 6a depicts chemical structures arranged in a reaction scheme, whichis an alternative to that shown in FIG. 4, for producing compoundswithin a third group of the family of preferred compounds;

FIG. 6b depicts chemical structures arranged in a reaction schemesimilar to that shown in FIG. 6a, except that the precursor compoundsutilized are chosen such that the compound produced has ameta-substitution rather than para-substitution on one phenyl ring andso that ethyl rather than methyl groups are present on the substituentmoiety of the meta-substituted phenyl ring;

FIGS. 7a and 7b depict chemical structures arranged in a reaction schemefor producing compounds within a fifth group of the family of preferredcompounds;

FIG. 8 is a graph illustrating the oxygen dissociation curves producedwhen a 5.4 millimolar solution of normal hemoglobin in the presence andabsence of selected compounds is tested at pH 7.4 using HEPES as thebuffer in a Hem-O-Scan oxygen dissociation analyzer;

FIG. 9 is a graph similar to FIG. 10 which illustrates oxygendissociation curves for whole human blood in the presence and absence ofselected compounds;

FIG. 10 is a graph similar to FIG. 8 where the oxygen dissociationcurves produced are for a 5.4 millimolar solution of normal hemoglobinin the presence and absence of particular compounds, including2,3-diphosphoglycerate which is the natural allosteric hemoglobineffector, are tested at pH 7.4 using HEPES as the buffer in a Hem-O-Scanoxygen dissociation analyzer;

FIG. 12 is a bar graph showing the percentage oxygen delivered by packedcells, fresh stored and in the presence of 2-[4-((((3,5-dimethylphenyl)amino)carbonyl)methyl)phenoxy]-2-methyl propionic acid (C₂₀ H₂₃ NO₄),respectively; and

FIGS. 12a-b depicts chemical structures in a reaction scheme used toproduce a phthalimide form of the compounds within the present inventionwhere the compounds were shown by a measured P value to allostericallymodify hemoglobin towards the low oxygen affinity state.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

Referring now to the drawings and, more particularly, to FIGS. 1a-cwhich illustrate the general structural formula of particularlypreferred compounds contemplated for use in the present invention andfirst and second subsets of the general structural formula,respectively. With reference to the general structural formula of FIG.1a the X and Z moieties may be CH₂, CO, NH or O, and the Y moiety may beCO or NH, with the caveat that the X, Y, and Z moieties are eachdifferent from one another. In addition, R₂₋₆ are either hydrogen,halogen, a substituted or unsubstituted C₁₋₃ alkyl group (up to threecarbons in length), or a C₁₋₃ ester or ether and these moieties may bethe same or different, or alkyl moieties of aliphatic or aromatic ringsincorporating two adjacent R₂₋₆ sites. The R₇₋₈ positions are hydrogen,halogen, methyl, or ethyl groups and these moieties may be the same ordifferent, or alkyl moieties as part of an aliphatic (e.g., cyclobutyl)ring connecting R₇ and R₈. The R₉ position is a hydrogen, halogen, C₁₋₃loweralkyl such as methyl, ethyl or propyl, or a salt cation such assodium, potassium, or ammonium.

In the first subset of compounds defined in FIG. 1b, X and Z may each beCH₂, NH, or O, with the caveat that when X is CH₂, Z is either NH or O,when X is NH, Z is either CH₂ or O, and when X is O, Z is NH or CH₂. Thefirst subset of compounds may conveniently be classified into fourgroupings as follows:

Group I: 2-[4-((aryl)acetamido)phenoxy]-2-methyl propionic acidcompounds having the general structural formula illustrated in FIG. 2C;

Group II: 2-[4-(((aryl)oxy)carbonyl)amino) phenoxy]-2-methyl propionicacid compounds having the general structural formula illustrated in FIG.3;

Group III: 2-[4-((((aryl)amino)carbonyl) methyl)phenoxy]-2-methylpropionic acid compounds having the general structural formulaillustrated in FIGS. 4 and 6a; and

Group IV: 2-[4-(((aryl)amino)carbonyl) oxy)phenoxy]-2-methyl propionicacid compounds having the general structural formula illustrated in FIG.5C. In the second subset of compounds defined in FIG. 1c, X and Z mayeach be CO or CH₂, with the caveat that when X is CO, Z is CH₂, and whenX is CH₂, Z is CO. The second subset of compounds may be convenientlydivided into two groupings as follows:

Group V: 2-[4-(((aryloyl)amino) methyl)phenoxy]-2-methyl propionic acidcompounds having the general structural formula illustrated in FIG. 7b.

Group VI: 2-[4-((((aryl)methyl)amino)carbonyl)phenoxy]-2-2-methylpropionic acid compounds which are thesubject matter of the co-pending U.S. patent application Ser. No.07/623,346 to Abraham et al. filed Dec. 7, 1990.

The R₂₋₉ substituents in FIGS. 1b-c are the same as those defined withreference to FIG. 1a. The synthesis of specific chemical compoundswithin the first five groups of compounds is provided in the followingexamples with reference to FIGS. 2-7. The synthesis of specific chemicalcompounds in the sixth group is explained in detail in co-pending U.S.patent application Ser. No. 07/623,346 to Abraham which has beenincorporated by reference. All compounds which were prepared werechecked by thin layer chromatography (TLC) for purity and the structureelucidation was based on NMR and IR spectroscopy and elemental analysis.

EXAMPLE 1

FIG. 2A illustrates a reaction scheme for preparing2-(4-aminophenoxy)-2-methyl propionic acid, a compound that is useful asa precursor in the preparation of Group I compounds. In accordance withthe scheme of FIG. 2A, 8 grams (g) (0.2 mol) of pulverized sodiumhydroxide is added to a suspension of 5.28 g (0.035 mol) ofp-acetaminophenol in 23 g (0.4 mol) of acetone. The reaction mixture isstirred at room temperature for 1/2 hour. Subsequently, 3.58 g (0.03mol) of chloroform is added dropwise over the course of 30 minutes. Thereaction mixture is stirred overnight at room temperature and acetone isremoved under vacuum. The residue is dissolved in water (10 ml),followed by acidification with 37% hydrochloric acid (HCl) to produce apale yellow precipitate of 2-(4-acetaminophenoxy)-2-methyl propionicacid (5 g, 60% yield), crystallized from methanol, mp 69°-71° C.

¹ H NMR (CD3OD) δ7.1(m,4H) ArH, 2.05 (s,3H), CH, 1.45, (s,6H) 2CH₃

1.18 g (0.005 mol) of the 2-(4-acetaminophenoxy)-2-methyl propionic acidis boiled in 10% KOH (60 ml) for 1/2 hour. The solution is then cooledand acidified with acetic acid to yield 0.6 g (62% yield) of2-(4-aminophenoxy)-2-methyl propionic acid as a yellowish white powder,mp 214°-16° C.

¹ H NMR: (DMSOd6+TMS) δ6.6 (m,4H)ArH, 1.35 (s,6H, 2CH₃ )

EXAMPLE 2

FIG. 2B illustrates another reaction scheme for preparing2-(4-aminophenoxy)-2-methyl propionic acid. In accordance with thescheme of FIG. 2B, 8 grams of potassium hydroxide is dissolved in 32 mlof water and the resultant KOH solution is admixed with 280 ml of 3%hydrogen peroxide. 11.3 g (0.058 mol) of 2-(4-cyanophenoxy)-2-methylpropionic acid is slowly added to the KOH/H₂ O₂ solution and thereaction mixture is stirred for about one hour until the exotherm andevolution of gas has ceased. The mixture is then cooled and acidifiedwith concentrated hydrochloric acid. The2-[4-(carboxamido)phenoxy]-2-methyl propionic acid product is obtainedas a white solid (9.8 g, 79% yield). The product is crystallized fromethanol to produce pure white crystals, mp 202°-4° C.

5.57 g (0.025 mol) of the 2-[4-(carboxamido)phenoxy]-2-methyl propionicacid is added gradually with stirring to 100 ml of an ice cold aqueoussolution containing 4.4 g (0.025 mol) of bromine and 11 g (0.25 mol) ofsodium hydroxide. The solution thus obtained is warmed at 75° C. for 1/2hour. After cooling, the solution is acidified with acetic acid givingthe desired 2-(4-aminophenoxy)-2-methyl propionic acid product as 4.0 g(81% yield) of a white precipitate, mp 214°-16° C. The compound isidentical with the product prepared in Example 1.

EXAMPLE 3

FIG. 2C illustrates a general reaction scheme for preparing the Group I2-[4-(arylacetamido)phenoxy]-2-methyl propionic acids. In accordancewith the illustrated scheme, 1 g (0.005 mol) of2-(4-aminophenoxy)-2-methyl propionic acid is dissolved with stirring in10 ml of water containing 0.41 g (0.1 mol) of NaOH. To this solution,0.79 g (0.005 mol) of phenyl acetyl chloride in 5 ml of tetrahydrofuran(THF) is gradually added over a period of about 15 minutes. After theaddition is complete the pH of the reaction mixture should be alkaline(if not a few drops of 2N NaOH is added to assure alkalinity). Thereaction mixture is continuously stirred for 1 hour. Thereafter, the THFis evaporated in vacuo, and the solution is then diluted with 5 ml waterand acidified with concentrated hydrochloric acid. The product isextracted with ethyl ether (2×20 ml), washed with water (3×20 ml), andthen dried over anhydrous MgSO₄. Upon addition of petroleum ether to theether solution, 0.9 g (56% yield) of the2-[4-(phenylacetamido)phenoxy]-2-methyl propionic acid productprecipitates as a pale brown solid, mp 173°-175° C.

¹ H NMR: (DMSOd6) 10 (s,1H, COOH), 7.5-6.7 (m, 9H, ArH), 3.55 (s, 2H,CH₂), 1.4 (s, 6H, 2CH₃)

Anal: C₁₈ H₁₉ NO₄

Calculated C 69.00 H; 6.07; N 4.47;

Found C 68.86 H; 6.14; N 4.42.

EXAMPLE 4

The procedure of Example 3 is followed as above, except that 0.005 molof 4-chlorophenyl acetyl chloride is substituted for the phenyl acetylchloride. In this case the product (57% yield) is2-[4-(p-chlorophenylacetamido)phenoxy]- 2-methyl propionic acid, mp168°-71° C.

¹ H NMR: (DMSOd6) δ10 (s, 1H, COOH), 7.6-6.7 (m, 8H, ArH), 3.6 (s, 2H,CHH₂), 1.4 (s, 6H, 2CH₃)

Anal: C₁₈ H₁₈ NO₄ Cl

Calculated C 62.15; H 5.17; N 4;02; Cl 10.12;

Found C 62.16; H 5.25; N 3.98 ; Cl 10.25.

The 4-chlorophenyl acetyl chloride for the foregoing synthesis isprepared by heating to reflux a suspension of 1 g (0.006 mol) of4-chlorophenyl acetic acid in 1.07 g (0.009 mol) of thionyl chloridewith stirring for 1 hour. After cooling, excess thionyl chloride isevaporated under vacuum to present the 4-chlorophenyl acetyl chlorideproduct as a yellow oil (1 g, 83% yield).

EXAMPLE 5

FIG. 3 illustrates a general reaction scheme for preparing the Group II2-[4-(((aryloxy)carbonyl)amino)phenoxy]-2-methyl propionic acids. Inaccordance with the illustrated scheme, a solution consisting of 0.15 g(0.001 mol) of phenyl chloroformate in 3 ml THF is gradually added to anice cold solution containing 0.3 g (0.001 mol) of 2-(4-aminophenoxy)-2-methyl propionic acid and 0.17 g (0.002 mol) of sodiumbicarbonate in 10 ml of water (10 ml). The reaction mixture is stirredfor 1/2 hour at 0° C., followed by stirring for 1 hour at roomtemperature. The THF is removed in vacuo and 10 ml of water is added.Then, the reaction mixture is acidified with concentrated hydrochloricacid and extracted with ethyl ether (2×20 ml). The ether solution iswashed with water (3×20 ml) and dried over anhydrous MgSO₄. The desiredproduct, 2-[4-((((phenyl)oxy)carbonyl) amino)phenoxy]-2-methyl propionicacid, is precipitated from the ether solution by addition of petroleumether as a white solid, 0.15 g (31% yield), mp 183°-5° C.

¹ H NMR: (DMSOd6) δ10 (s, 1H, COOH), 7.55-6.75 (m, 9H, ArH), 1.4 (s, 6H,2CH₃)

Anal: C₁₇ H₁₇ O₅ N

Calculated C 64.7; H 5.39; N 4.44;

Found C 64.65 H 5.45; N 4.43.

EXAMPLE 6

The procedure for Example 5 is followed as above except that 0.001 molof 4-chlorophenyl chloroformate is substituted for the phenylchloroformate. In this case the2-[4-((((p-chlorophenyl)oxy)carbonyl)amino)phenoxy]-2-methyl propionicacid product is obtained as a white precipitate, 0.15 g (28% yield), mp179°-82° C.

¹ H NMR: (DMSOd₆ +TMS) δ7.6-6.8 (m, 8H, ArH), 1.4 (s, 6H, 2CH₃)

Anal: C₁₇ H₁₆ O₅ NCl

Calculated C 58.36; H 4.57; Cl 10.15;

Found C 58.16; H 4.68; Cl 10.35.

EXAMPLE 7

FIG. 4 illustrates a general reaction scheme for preparing the Group IIIcompounds of the invention. In accordance with the illustrated scheme,5.2 g (34 mmol) of 4-hydroxyphenylacetic acid (HPAA) is heated to refluxwith an excess of thionyl chloride (SOCl,) for 1/2 hour. The reactionmixture is then cooled and excess SOCl₂ is removed under vacuum. Theresidue is reacted for 2 hours with 6.3 g (68 mmol) of aniline in 50 mlof refluxing xylene. The reaction mixture is then cooled, washed withdilute HCl, water and brine and extracted with aqueous 2N NaOH. Thecombined alkali layer is washed with ether, cooled and acidified toprovide 7 g of solid N-phenyl-4-hydroxybenzyl amide (C₁₄ H₁₂ NO₂) as anintermediate product (90% yield), mp 138° C. The intermediate product isrecrystallized from a 1:2 mixture of acetone and petroleum ether and a1.13 g (5 mmol) portion is O-alkylated for 12 hours using the procedureof Example 1 with 20 ml acetone, 2.75 g NaOH and 1.25 ml CHCl₃. Thefinal pro<xct is 2-[4-((((phenyl)amino)carbonyl)methyl)phenoxy]-2-methyl propionic acid (C₁₈ H₁₉ NO₄), 1.2 g (76%yield), mp 198° C.

EXAMPLE 8

The procedure of Example 7 is repeated using 8.6 g (68 mmol) of4-chloroaniline rather than the aniline. In this case, the intermediateproduct is N-(4-chlorophenyl)-4-hydroxy benzylamide (C₁₄ H₁₂ ClNO₂), 7.5g (84% yield), mp 163° C. 1.3 g of the intermediate product isO-alkylated to produce 2-[4-((((4-chlorophenyl)amino)carbonyl)methyl)phenoxy]-2-methyl propionic acid (C₁₈ H₁₈ ClNO₄), 0.86 g (50% yield), mp196° C.

EXAMPLE 9

The procedure of Example 7 is repeated using 2.6 g (17 mmol) of the HPAAand using 5.67 g (35 mmol) of 3,4-dichloroaniline rather than aniline.In this case, the intermediate product isN-(3,4-dichlorophenyl-4-hydroxy benzylamide (C₁₄ H₁₁ Cl₂ NO₂). 1.48 g (5mmol) of the intermediate is O-alkylated to produce2-[4-(((3,4-dichlorophenyl)amino) carbonyl)methyl)phenoxy]-2-methylpropionic acid (C₁₈ H₁₇ Cl₂ NO₄), 0.76 g (40% yield), mp 174° C.

EXAMPLE 10

The procedure of Example 7 is repeated using 2.6 (17 mmol) of the HPAAand using 5.7 g (35 mmol) of 3,5-dichloroaniline rather than aniline. Inthis case, the intermediate product is N-(3,5-dichlorophenyl-4-hydroxybenzylamide (C₁₄ H₁₁ Cl₂ NO₂). 1.48 g (5 mmol) of the intermediate isO-alkylated to produce 2-[4-((((3,5-dichlorophenyl)amino)carbonyl)methyl)phenoxy]-2-methyl propionic acid (C₁₈ H₁₇ Cl₂ NO₄), 0.8g (42% yield), mp 138° C.

EXAMPLE 11

The procedure of Example 7 is repeated using 0.95 g (6 mmol) of theHPAA, 2.6 g (12 mmol) of 3,4,5-trichloroaniline rather than aniline, and25 ml of refluxing xylene. In this case, the intermediate product isN-(3,4,5-trichlorophenyl)-4-hydroxy benzylamide. 0.50 g (1.5 mmol) ofthe intermediate product is O-alkylated using 10 ml acetone, 0.82 g NaOHand 0.37 ml CHCl₃ to produce2-[4-((((3,4,5-trichlorophenyl)amino)carbonyl) methyl)phenoxy]-2-methylpropionic acid (C₁₈ H₁₆ CL₃ NO₄), 0.27 g (43% yield), mp 160° C.

EXAMPLE 12

The procedure of Example 7 is repeated using 5.04 g (32 mmol) of theHPAA, 6 ml (64 mmol) of 4-fluoroaniline rather than aniline, and 25 mlof refluxing xylene. In this case, the intermediate product isN-(4-fluorophenyl)-4-hydroxybenzylamide. 1.22 g (5 mmol) of theintermediate product is 0-alkylated to produce2-[4-((((4-fluorophenyl)amino)carbonyl)methyl)phenoxy]-2-methylpropionic acid (C₁₈ H₁₈ FNO₄), 0.74 g (45% yield), mp 198° C.

EXAMPLE 13

The procedure of Example 7 is repeated using 5.04 (32 mmol) of the HPAA,8.05 ml (64 mmol) of 4-trifluoromethylaniline rather than aniline, and25 ml of refluxing xylene. In this case, the intermediate product isN-(4-trifluoromethylphenyl)-4-hydroxy benzylamide. 1.5 g (5 mmol) of theintermediate is used to produce2-[4-((((4-trifluoromethylphenyl)amino)carbonyl)methyl)phenoxy]-2-methyl propionic acid (C₁₉ H₁₈ F₃ NO₄), 0.85 g (44% yield),mp 197° C.

EXAMPLE 14

The procedure of Example 7 is repeated using 5 04 (32 mmol) of the HPAA,8 g (65 mmol) of 4-methyl aniline rather than aniline, and using 25 mlof refluxing xylene. In this case the intermediate product isN-(4-methylphenyl)-4-hydroxy benzylamide. 1.2 g (5 mmol) of theintermediate is used to produce 2-[4-((((4-methylphenyl)amino)carbonyl)methyl) phenoxy]-2-methyl propionic acid (C₁₉ H₂₁ NO₄), 0.98 g(65% yield), mp 164° C.

EXAMPLE 15

The procedure of Example 7 is repeated using 3.26 (21 mmol) of the HPAA,5.3 ml (42 mmol) of 3,5-dimethyl aniline rather than aniline, and 25 mlof refluxing xylene. In this case the intermediate product isN-(3,5-dimethylphenyl)-4-hydroxy benzylamide. 1.27 g (5 mmol) of theintermediate is used to produce 2-[4 ((((3,5-dimethylphenyl)amino)carbonyl)methyl)phenoxy]-2-methyl propionic acid (C₂₀ H₂₃ No₄),1.15 g (68% yield), mp 85° C. Alternatively, the procedure outlined inthe German Patent Application 2,432,560, which is herein incorporated byreference, can be followed to produce the compound of this Example 15.

EXAMPLE 16

The procedure of Example 7 is repeated using 5.04 (32 mmol) of the HPAA,10 ml (64 mmol) of 4-isopropyl aniline rather than aniline, and using 5ml of refluxing xylene. In this case the intermediate product isN-(4-isopropylphenyl)-4-hydroxybenzylamide. 1.34 g (5 mmol) of thesemisolid, thick viscous liquid intermediate is used to prepare2-[4-((((4-isopropylphenyl)amino)carbonyl) methyl)phenoxy]-2-methylpropionic acid (C₂₁ H₂₅ NO₄), 1.1 g (61% yield), mp 141° C.

EXAMPLE 17

With reference to FIGS. 5A, 5B and 5C, a scheme is illustrated forpreparing Group IV compounds. In accordance with FIG. 5A, aniline oraniline derivatives may be reacted with phosgene to obtain the carbamoylchloride. In accordance with FIG. 5B, hydroquinone may be monoacetylatedusing acetic anhydride. The product is then O-alkylated using acetone,CHCl₃ and KOH and then hydrolyzed using a base. The products of thereactions of FIGS. 5A and 5B may then be reacted according to thereaction scheme of FIG. 5C to produce the Group IV2-[4-(((arylamino)carbonyl)oxy)phenoxy)]-2-methyl propionic acids.

EXAMPLE 18

As an alternative to the reaction scheme described in Example 7 andshown in FIG. 4, the Group III compounds may be prepared according tothe scheme shown in FIG. 6a. 5.2 g (32 mmol) of HPAA, 6.3 g (68 mmol) ofaniline, and 25 ml of mesitylene are heated to reflux. 0.74 g (8mmol) ofphosphorous pentachloride is added to the refluxing mixture and thereflux is continued for an additional two hours. The reaction mixture issubsequently cooled, washed with dilute HCl, water and brine, andextracted with aqueous 2N sodium hydroxide NaOH. The combined alkalilayer is washed with ether, cooled and acidified to provide 7 g (90%yield) of solid N-phenyl-4-hydroxybenzyl amide (C₁₄ H₁₂ NO₂) as anintermediate product, mp 138°. The intermediate product isrecrystallized from a 1:2 mixture of acetone:petroleum ether and a 1.13g (5 mmol) portion is O-alkylated. 1.6 g (30 mmol) of pulverized sodiumhydroxide is added to a solution of N-phenyl-4-hydroxybenzamide (1.13g,5mmol) in 20 ml of acetone. The reaction mixture is stirred overnight atroom temperature and acetone is removed under vacuum. The residue isdissolved in 10 ml of water and acidified with 2N HCl to produce a paleyellow solid. The solid is separated, dissolved in methanol,charcoalated, and solvent evaporated to provide2-[4-((((phenyl)amino)carbonyl)methyl) phenoxy]-2-methyl propionic acid(C₁₈ H₁₉ NO₄), 1.2 g (76% yield), mp 198° C. The last step in theprocedure shown in FIG. 6a is the conversion of the acid to the sodiumsalt via its reaction with sodium bicarbonate. Similar reactions withother salt cations such as potassium and ammonium or reactions to formesters can also be performed.

EXAMPLE 19

FIG. 6b presents a similar reaction scheme to FIG. 6a, except that 3-rather than 4-hydroxyphenylacetic acid (HPAA) is used as the precursormaterial so that the final compound has a meta rather than a parasubstitution. In addition, rather than reacting with acetone (dimethylketone) a diethyl ketone is used to position ethyl, rather than methyl,moieties in the group substituted on one of the phenyl rings. Byexample, 1.5g (10mmol) 3-HPAA and 2.6g (20 mmol) 4-chloroaniline in 20ml of mesitylene was heated to reflux. Then 0.33g (2.55 mmol) PCl₅solution was then slowly added to the above refluxing solution and therefluxing was continued for two hours. The reaction mixture was thencooled and then worked up as described above to yield 2.2 g (90% yield)of 3-[((4-chloroanilino) carbonyl)methyl]phenol. As described above,chloroform (0.8 ml) was added to a stirred and ice cooled mixture of1.23 grams of 3-[((4-chloroanilino)carbonyl)methyl]phenol and 1.6 g NaOHin 15 ml of acetone. The reaction mixture was allowed to warm to roomtemperature and stirring continued for an additional 10 hours. The usualwork-up yielded 2-[3-(((4-chloroanalino)carbonyl)methyl)]phenoxy]-2-methylpropionic acid as a low temperature meltingsticky solid (C,H,Cl,N analysis yielded (C₁₈ H₁₈ ClNO₄); NMR δPPM: 1.42(6H, s, CH₃), 3.61 (2H, s, benzylic CH₂), and 6.6-7.75 (8H, m, aromaticH)). However, rather than using acetone as the reaction solvent,diethylketone can be used in the same manner as described above to yieldthe butanoic acid (as opposed to propanoic acid) structure shown in FIG.6b.

EXAMPLE 20

With reference to FIG. 7a, a general reaction scheme for preparing2-[4-(aminomethyl)phenoxy]-2-methyl propionic acid, a compound that isuseful as a precursor to the preparation of the Group V compounds, ispresented. In accordance with the illustrated scheme,2-[4-cyanophenoxy]-2-methyl propionic acid (2 g, 9 mmol), prepared asdescribed in Example 2, and 75 ml of ethanol were placed in a 250 mlParr hydrogenation bottle. The solution was acidified with concentratedhydrochloric acid (3 ml), then 10% palladium on activated charcoal (0.2g, 10% wt) was added to the mixture. The reaction mixture was placed ona Parr hydrogenator apparatus at 45 psi of hydrogen pressure and shakenfor a period of two hours. The mixture was filtered to remove thecatalyst, and the filtrated concentrated under vacuum. Addition of etherprecipitated hydrochloride salt of the desired product as white, shinycrystals (2.1 g, 87%).

EXAMPLE 21

FIG. 7B illustrates a general reaction scheme for preparing the Group Vcompounds used in the present invention. In accordance with theillustration, a solution of benzoyl chloride (0.14 g, 1 mmol) in THF (3ml) was added over a 15 minute period to a stirred solution of2-[4-(aminomethyl)phenoxy]-2-methylpropionic acid (0.24 g, 1 mmol) andNaOH (0.08 g, 2 mmol) in 10 ml of water. After the addition of thebenzoyl chloride was completed, the reaction mixture was stirred for 1hour at room temperature. THF was evaporated in vacuo. Acidification ofthe residue provided the desired compound as an oil which was extractedwith ether. The organic layer was washed with water, brine, and airedover anhydrous MgSO₄. Subsequent addition of petroleum etherprecipitated 2-[4-(benzoylamino)methyl)phenoxy]-2-methyl propionic acid(C₁₈ H₁₉ NO₄) as a white solid (0.15 g, 48%) mp 176°-179° C.

NMR: (DMSO-d₆) δ 1.45 (6H, s, 2CHH₃), 4.4 (2H, d, CH₂), 6.8-7.2 (4H, dd,J=9 Hz, aromatic H, 7.4-8 (5 H, m, aromatic H), 9, 1 H, br t, NH).

EXAMPLE 22

The procedure of Example 21 is repeated using 2-chlorobenzoyl chloride(1 mmol) rather than benzoyl chloride. In this case, the product (58%yield) is 2[4-(((2-chlorobenzoyl)amino)methyl)phenoxy]-2-methylpropionic acid (C₁₈ H₁₈ ClNO₄) mp 135°- 137° C.

EXAMPLE 23

The procedure of Example 21 is repeated, except that 1 mmol of3-chlorobenzoyl chloride is substituted for benzoyl chloride. In thiscase, the product (53% yield) is2-[4-(((3-chlorobenzoyl)amino)methyl)phenoxy]-2-methyl propionic acid(C₁₈ H₁₈ ClNO₄) mp 145°-146° C.

EXAMPLE 24

The procedure of Example 21 is repeated, except that 1 mmol of4-chlorobenzoyl chloride is substituted for benzoyl chloride. In thiscase, the product (63% yield) is2-[4-(((4-chlorobenzoyl)amino)methyl)phenoxy]-2-methyl propionic acid(C₁₈ H₁₈ ClNO₄) mp 186°-189° C.

EXAMPLE 25

The procedure of Example 21 is repeated, except that 1 mmol of3,4-dichlorobenzoyl chloride is substituted for benzoyl chloride. Inthis case, the product (57% yield) is2-[4-(((3,4-dichlorobenzoyl)amino)methyl)phenoxy]-2-methyl propionicacid (C₁₈ H₁₇ Cl₂ NO₄) mp 186°-189° C.

EXAMPLE 26

The procedure of Example 20 is repeated, except that 1 mmol of3,5-dichlorobenzoyl chloride is substituted for benzoyl chloride. Inthis case, the product (43% yield) is2-[4-(((3,5-dichlorobenzoyl)amino)methyl)phenoxy]-2-methyl propionicacid (C₁₈ H₁₇ Cl₂ NO₄) mp 110°-113° C.

EXAMPLE 27

The procedure of Example 20 is repeated, except that 1 mmol of3,4,5-trichlorobenzoyl chloride is substituted for benzoyl chloride. Inthis case, the product is2-[4-(((3,4,5-trichlorobenzoyl)amino)methyl)phenoxy]-2-methyl propionicacid (C₁₈ H₁₆ Cl₃ NO₄) mp 151°-152° C.

Examples 1 through 27 outline the synthesis procedures for producingseveral compounds within the family of compounds defined by the generalstructural formula of FIG. 1a. Specifically, Examples 1-19 disclosesynthesis procedures for Groups 1-4 compounds within the subset definedby the structural formula of FIG. 1b and Examples 20-27 disclosesynthesis procedures for Group 5 compounds within the subset defined bythe structural formula of FIG. 1c. The co-pending U.S. patentapplication Ser. No. 07/623,346 to Abraham et al. filed Dec. 7, 1990,describes the synthesis procedures for Group 6 compounds within thesubset defined by the structural formula of FIG. 1c. It should beunderstood that other compounds within the family of compounds used inthe present invention can easily be synthesized by changing the startingmaterials. All compounds within the family would have a similar mode ofbinding and would, therefore, all should have the effect of shifting theallosteric equilibrium of hemoglobin towards favoring the low affinity"T" state.

The broad family of compounds contemplated for use in this inventionincludes compounds defined by the formula: ##STR3## where R₂ is asubstituted or unsubstituted aromatic such as a phenyl, naphthyl, orindanyl, or hetrocyclic aromatic, or a substituted or unsubstitutedalkyl ring compound, such as a cyclohexyl or adamantyl, or a substitutedor unsubstituted phthalimide compound where X is a carboxyl, Y is anitrogen and R₂ completes the phthalimide compound by being bonded toboth X and Y, and where X, Y, and Z are CH₂, NH, CO, O or N with thecaveat that the X, Y, and Z moieties are each different from oneanother, and where R₁ has the formula: ##STR4## where R₁ can beconnected to any position on the phenyl ring and R₃ and R₄ are hydrogen,halogen, methyl, or ethyl groups and these moieties may be the same ordifferent, or alkyl moieties as part of an aliphatic ring connecting R₃and R₄, and R₅ is a hydrogen, loweralkyl such as methyl, ethyl orpropyl, or a salt cation such as sodium, potassium, or ammonium. To thisend, compounds have having a naphthyl, adamantyl, or indanyl group at R₁instead of the substituted phenyl like that shown in FIG. 1a have beenprepared using substantially the same synthetic routes as describedabove. In addition, compounds having a phthalimide-like structure havealso been synthesized as shown in FIGS. 12a-b and described below inEXAMPLE 28.

EXAMPLE 28

FIGS. 12a and 12b show alternative synthesis routes for preparing2-[4-((phthalamido)N-methyl)phenoxy)-2-methyl proprionic acid. Phthalicanhydride (0.75 g; 5 mmol) and 2[4-((amino)methyl)phenoxy]-2-methylproprionic acid (see FIG. 2B) were refluxed in 25 ml of toluene in thepresence of 1 ml triethylamine. Water was removed azeotropically. Afterfour hours of refluxing, the reaction mixture was cooled, toluene wasseparated, and the above-described work up was provide to yield acrystalline white residue (90% yield; mp 149° C.; NMRδ ppm: 1.46(6H, s,CH₂), 4.65(2H, s, CH₂), 6.75 and 7.2 (4H, d, J=6 Hz, aromatic H of apara-substituted ring), and 7.85 (4H, s, aromatic H of a phthalimideunit).

To test the compounds of the invention for physiological activity, humanblood was obtained from the Central Blood Bank, Richmond, Virginia. Theextraction, chromatography, and characterization of isolated hemoglobinmethods used by the inventors were identical to those described by Dozyand Huisman in J. of Chromatography, Vol 32, (1968) pp. 723 and in TheChromatography of Hemoglobin, H. J. Schroeder and D. H. J. Huisman, Ed.Marcel Dekker Inc. N.Y. (1980) which are herein incorporated byreference. The purity of normal hemoglobin (HbA) was determined by gelelectrophoresis, using a Gelman semimicroelectrophoresis chamber. Theconcentration of hemoglobin was determined according to thecyanmethemoglobin method described in Zijlstra, Clin. Chem. Acta., Vol5, pp. 719-726 (1960), and Zijlstra and Van Kamper, J. Clin. Chem. Clin.Biochem., Vol. 19, p. 521 (1981) which are herein incorporate byreference. All purified hemoglobin solutions were stored in liquidnitrogen. The reagents and buffers were purchased from the followingsources: Fischer Scientific, Sigma Chemical Company, and Pharmacia andResearch Chemicals, Inc.

Oxygen equilibrium curves were determined on an AMINCO™ HEM-O-SCANoxygen dissociation analyzer available from Travenol Laboratories. HbAwas prepared as follows: 20 ml of whole blood from a nonsmoking donor(blood bank, Richmond, Va.) was drawn into a heparinized vacutainer. Theblood was immediately packed in ice (to prevent MetHb formation) andthen centrifuged (10 minutes at 2500 rpm) to separate the plasma andbuffy coat from the packed erythrocytes. After centrifugation wascompleted, the plasma and buffy coat were removed by aspiration and thecells washed three times with 0.9% NaCl containing 40 mg ofethylenediaminetetraacetic acid (EDTA) per liter and then once with 1.0%NaCl containing 40 mg of EDTA/L. The cells were lysed by the addition ofone to two volumes of deionized water containing 40 mg of EDTA/L. Themixture was allowed to stand for 30 minutes with occasional mixingbefore being centrifuged for two hours at 10,000 rpms at 4° C. for twohours to remove the remaining cell stroma. The supernatant was furtherpurified by either gel filtration with Sephadex G-25 or dialysis againstpH 8.6 tris buffer (50 mM, containing 40 mg. of EDTA/L). The sodiumchloride free hemoglobin solution was chromatographed on DEAE-Sephacelion-exchange resin (Sigma) preequilibrated with Tris buffer (pH 8.6, 50mM, containing 40 mg of EDTA/L), the HbA fraction was then eluted withpH 8.4 Tris buffer. The pure HbA fraction (identified byelectrophoresis) was concentrated using a Schleicher and Schuellcollodion bag apparatus (Schleicher and Schuell, Inc.) with HEPES buffer(150 mM, pH 7.4) as the exchange buffer. The hemoglobin concentrationwas then determined using the above-noted cyanomethemoglobin method. Thehemoglobin concentration at this point was usually found to be around 35g % or approximately 5.5 mM. Less than 5% methemoglobin was noted evenafter several days at 4° C.

All compounds were mixed with one equivalent of sodium bicarbonate(NaHCO₃) (this process converts the carboxylic acid moiety to a sodiumsalt; see FIG. 6a), then dissolved in the HEPES buffer to give 20 mMsolutions. Just prior to running the oxygen equilibrium curve, thehemoglobin and the drug were mixed in a 1:1 ratio (50 μl of hemoglobinplus 50 μl of drug) to give 2.75 mM hemoglobin with a drug concentrationof 10 mM. The control was prepared by the addition of 50 μl ofhemoglobin to 50 μl of the HEPES buffer.

Table 1 presents the measured P₅₀ value, the P₅₀ control value, and theratio of the measured P₅₀ value to the control (P₅₀ /P₅₀ c) for normalhemoglobin treated with several synthesized compounds.

                                      TABLE 1                                     __________________________________________________________________________    Comp.                          P.sub.50                                       No.  R.sub.2                                                                         R.sub.3                                                                           R.sub.4                                                                            R.sub.5                                                                          R.sub.6                                                                         X  Y  Z   (Control)                                                                          P.sub.50                                                                         P.sub.50 /P.sub.50 C                   __________________________________________________________________________     1   H H   H    H  H CO NH CH.sub.2                                                                          18   35 1.94                                    2   Cl                                                                              H   H    H  H CO NH CH.sub.2                                                                          18   27.5                                                                             1.52                                    3   H Cl  H    H  H CO NH CH.sub.2                                                                          18   37.5                                                                             2.08                                    4   H H   Cl   H  H CO NH CH.sub.2                                                                          19   48 2.52                                    5   H Cl  Cl   H  H CO NH CH.sub.2                                                                          18   40.5                                                                             2.25                                    6   H Cl  H    Cl H CO NH CH.sub.2                                                                          18   47 2.60                                    7   H Cl  Cl   Cl H CO NH CH.sub.2                                                                          19   40 2.10                                    8   H H   H    H  H CH.sub.2                                                                         CO NH  19   35 1.73                                    9   H Cl  H    H  H CH.sub.2                                                                         CO NH  18   44 2.44                                   10   H H   Cl   H  H CH.sub.2                                                                         CO NH  19   44 2.31                                   11   H H   F    H  H CH.sub.2                                                                         CO NH  18   35 1.94                                   12   H H   CH.sub.3                                                                           H  H CH.sub.2                                                                         CO NH  18   45 2.50                                   13   H H   CF.sub.3                                                                           H  H CH.sub.2                                                                         CO NH  18   42 2.33                                   14   H H   OMe  H  H CH.sub.2                                                                         CO NH  18   38 2.11                                   15   H Cl  Cl   H  H CH.sub.2                                                                         CO NH  18   50 2.77                                   15   H Me  H    Me H CH.sub.2                                                                         CO NH  18   52 2.88                                   16   H H   H    H  H O  CO NH  18   26.5                                                                             1.47                                   17   H H   Cl   H  H O  CO NH  19   34 1.78                                   18   H H   H    H  H NH CO CH.sub.2                                                                          19   54 2.84                                   19   H H   Cl   H  H NH CO CH.sub.2                                                                          19   54 2.84                                   20   H Cl  Cl   H  H NH CO CH.sub.2                                                                          18   65 3.61                                   21   H Cl  H    Cl H NH CO CH.sub.2                                                                          19   83 4.36                                   22   H Cl  Cl   Cl H NH CO CH.sub.2                                                                          19   63 3.30                                   23   H H   F    H  H NH CO CH.sub.2                                                                          18   45 2.50                                   24   H H   CF.sub.3                                                                           H  H NH CO CH.sub.2                                                                          18   44 2.44                                   25   H H   CH.sub.3                                                                           H  H NH CO CH.sub.2                                                                          18   49 2.72                                   26   H CH.sub.3                                                                          H    CH.sub.3                                                                         H NH CO CH.sub.2                                                                          19   75 3.94                                   27   Cl                                                                              H   H    H  Cl                                                                              NH CO CH.sub.2                                                                          18   34 1.89                                   28   H Me  Me   H  H NH CO CH.sub.2                                                                          18   62 3.41                                   29   --                                                                              NAPTHYL  -- --                                                                              NH CO CH.sub.2                                                                          18   58 3.20                                   30   H PROPYL   H  H NH CO CH.sub.2                                                                          18   64 3.56                                   27   H Cl  H    H  H NH CO CH.sub.2                                                                          18   61 3.40                                   28   H H   Cl   H  H CH.sub.2                                                                         NH CO  18   27 1.50                                   29   H H   CH.sub.3                                                                           H  H CH.sub.2                                                                         NH CO  19   28 1.47                                   30   H Cl  Cl   H  H CH.sub.2                                                                         NH CO  18   28 1.56                                   31   H H   H    H  H CH.sub.2                                                                         NH CO  19   22 1.16                                   32   --                                                                              INDANYL  -- --                                                                              NH CO CH.sub.2                                                                          18   64 3.56                                   33   --                                                                              ADAMANTYL                                                                              -- --                                                                              NH CO CH.sub.2                                                                          18   32 1.78                                   34 BZF                                                                             H H   Cl   H  H CO NH (CH.sub.2).sub.2                                                                  18   33 1.83                                   __________________________________________________________________________

It is noted that the P₅₀ control value is less than for normalhemoglobin under physiological conditions (e.g., 26.5) because here theP₅₀ was made on hemoglobin in solution (outside the red blood cells.Each hemoglobin sample treated with one of the compounds falling withinthe family defined by this invention had a P₅₀ drug value which wasgreater than the P₅₀ control. This response indicates that theallosteric equilibrium for hemoglobin has been shifted towards favoringthe low oxygen affinity "T" state of hemoglobin due to the presence ofthe compounds. At the bottom of Table 1, a row (34) is presented forbezafibrate (BZF), a known "right-shifting" allosteric hemoglobinmodifier. As with all the newly discovered "right-shifting" allosterichemoglobin modifiers, the hemoglobin treated with BZF had a higher P₅₀than the P₅₀ for the control. Table 1 shows the varying R₂₋₆ moietiesfor the substituted phenyl compounds tested, and when a compound whichdid not have a substituted phenyl, the name of the compound is writtenacross R₂₋₆ (e.g., naphthyl, adamantyl, indanyl). The R₇₋₈ moieties weremethyl groups for each compound tested and the R₉ moiety was a sodiumcation for each compound tested (derived from the NaHCO₃ treatment priorto testing). Because other compounds within the family would have asimilar mode of binding (e.g., those with different R₂₋₉ moieties),their effect on the P₅₀ value can be expected to be the same. Thephthalimide structure defined by FIGS. 12a-b and Example 29 had a meanP₅₀ value (e.g., P₅₀ Drug/P₅₀ Control) of 1.08 indicating the allostericequilibrium for hemoglobin had been shifted towards favoring the lowoxygen affinity "T" state of hemoglobin by the phthalimide compound.

Table 2 shows the effect some of the compounds have on the oxygendissociation of normal hemoglobin in intact human red blood cells(RBCs).

                  TABLE 2                                                         ______________________________________                                        NO.       R.sub.2                                                                             R.sub.3                                                                              R.sub.4                                                                            R.sub.5                                                                            R.sub.6                                                                           x     z    P.sub.50                      ______________________________________                                        CONTROL   --    --     --   --   --  --    --   27                            21        H     CH.sub.3                                                                             H    CH.sub.3                                                                           H   NH    CH.sub.2                                                                           83                            22        H     Cl     H    Cl   H   NH    CH.sub.2                                                                           87                            23        H     CH.sub.3                                                                             H    CH.sub.3                                                                           H   NH    CH.sub.2                                                                           76                            24        H     CH.sub.3                                                                             H    CH.sub.3                                                                           H   NH    CH.sub.2                                                                           68                            ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        PACKED RBC  P.sub.50 IN PRESENCE OF RSR-13                                    DAY OLD     0 mM        1 mM      2 mM                                        ______________________________________                                        FRESH       38          --        --                                          40          32          39        45                                          50          33          39        45                                          60          34          40        47                                          70          35          39        50                                          ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        PACKED RBC  P.sub.50 UPON ADDITION OF RSR-13                                  DAY OLD     0 mM        1 mM      2 mM                                        ______________________________________                                        FRESH       38          --        --                                          40          32          38        42                                          50          31          38        45                                          60          34          39        46                                          ______________________________________                                    

The first entry provides the P₅₀ value obtained for a control of humanRBCs alone. The next two entries provide the P₅₀ values when the RBCsare mixed together with a 10 millimolar (mM) solution of the sodium saltof either2-[4-((((3,5-diclorophenyl)amino)carbonyl)methyl)phenoxy]-2-methylpropionic acid (C₁₈ H₁₇ Cl₂ NO₄) (discussed in Example 10) or2-[4((((3,5-dimethylphenyl)amino)carbonyl)methyl)phenoxy]-2-methylpropionic acid (C₂₀ H₂₃ NO₄) (discussed in Example 15), respectively.Note that the P₅₀ values for the hemoglobin in intact RBCs treated withthe compounds is much greater than the P₅₀ value for untreatedhemoglobin under physiological conditions (e.g., the control of 27). Inaddition, it was determined that the P₅₀ value was raised from 27 to 31in the presence of 1 mM2-[4((((3,5-dimethylphenyl)amino)carbonyl)methyl)phenoxy]-2-methylpropionic acid and to 42 in the presence of 2 mM2-[4((((3,5-dimethylphenyl)amino)carbonyl)methyl)phenoxy]-2-methylpropionic acid. This data establishes the permeability of the of thecompounds to the cell membrane and that serum albumin does not interferewith the drug's influence on the oxygen dissociation curve ofhemoglobin. Entries 23 and 24 in Table 2 provide the P₅₀ values forintact RBCs treated with 10 mM of the same two compounds used in entries21 and 22, respectively, except that the RBCs were washed with a 240fold excess of 0.9% saline. The relatively slight drop in the P₅₀ valueafter the saline wash, which represents a high retention of allostericeffect, shows that the compounds used in the present invention have highbinding affinity for hemoglobin.

FIG. 8 is a graph illustrating the oxygen dissociation curves producedwhen a 5.4 millimolar solution of normal hemoglobin is tested at pH 7.4using HEPES as the buffer in a Hem-O-Scan oxygen dissociation analyzer.As described above, the P₅₀ values reported in Table 1 were determinedfrom curves like those shown in FIG. 8. With particular reference toFIG. 8, the percent oxygen saturation (SO₂ on the vertical axis) isplotted against the partial pressure of oxygen (PO₂ on the horizontalaxis). Curve number 1 shows the oxygen dissociation curve (ODC) in theabsence of an allosteric modifying agent. Curve number 2 shows the ODChas been shifted to the right when 10 mM bezafibrate (a known rightshifting agent) solubilized with an equimolar amount of NaHCO₃ is addedto the hemoglobin. It should be noted that as the curve is right shiftedto a lower oxygen affinity state, the P₅₀ value increases. Curve number3 shows the right shift caused by adding a 10 mM concentration of2-[4-((((4-chlorophenyl)amino)carbonyl)methyl)phenoxy]-2-methylpropionic acid (C₁₈ H₁₈ ClNO₄) (described in Example 8 above) to thehemoglobin. Curve number 4 shows the right shift caused by adding a 10mM concentration of2-[4-((((3,5-dimethylphenyl)amino)carbonyl)methyl)phenoxy]-2-methylpropionic acid (C₂₀ H₂₃ NO₄) (described in Example 15) to thehemoglobin. Finally, curve number 5 shows the right shift caused byadding a 10 mM concentration of2-[4-((((3,5-dichlorophenyl)amino)carbonyl)methyl)phenoxy]-2methylpropionic acid (C₁₈ H₁₇ Cl₂ NO₄) (described in Example 10) to thehemoglobin. The right shifting effect shown in FIG. 8 indicates thecompounds may be used to lower the oxygen affinity of hemoglobin.

FIG. 9 illustrates the effect of particular compounds on the ODC ofwhole human blood. Like FIG. 8, the percent oxygen saturation is plottedagainst the partial pressure of oxygen. As described above, the P₅₀values reported in Table 2 were determined from curves like those shownin FIG. 9. For these curves, 50 μl of whole human blood was mixed with a50 μl solution of the test compound in HEPES buffer at pH 7.4. Curvenumber 1 shows the ODC of hemoglobin in unreacted whole blood. Curves 2and 3 respectively illustrate the right shifting effect of the salts ofa 10 mM concentration of2-[4-((((3,5-dimethylphenyl)amino)carbonyl)methyl)phenoxy]-2-methylpropionic acid (C₂₀ H₂₃ NO₄) (described in Example 15) or a 10 mMconcentration of2-[4-((((3,5-dichlorophenyl)amino)carbonyl)methyl)phenoxy]-2-methylpropionic acid (C₁₈ H₁₇ Cl₂ NO₄) (described in Example 10) on hemoglobinin whole blood.

FIG. 10 shows ODC curves of human hemoglobin (5.4 mM) in HEPES buffer atpH 7.4 which were made in a manner similar to that described inconjunction with FIG. 8. Like FIGS. 8 and 9, the percent oxygensaturation is plotted against the partial pressure of oxygen. Curvenumber 1 shows ODC of human hemoglobin in the absence of any allostericmodifying agent. Curves 2 and 3 show the right shifting effect of 1 mMand 10 mM concentrations of2-[4-((((3,5-dimethylphenyl)amino)carbonyl)methyl)phenoxy]-2-methylpropionic acid (C₂₀ H₂₃ NO₄) (described in Example 15) on humanhemoglobin. Hence, this compound forces hemoglobin to a lower oxygenaffinity state. Curve number 4 shows the right shifting effect of 2.5 mM2,3-diphosphoglycerate (2,3-DPG), which is a natural allosterichemoglobin effector. Curve number 5 shows the combined effect of twoeffectors, e.g., 1 mM2-[4-((((3,5-dimethylphenyl)amino)carbonyl)methyl)phenoxy]-2-methylpropionic acid and 2.5 mM 2,3-DPG, is greater than either effectoralone. The synergistic effect may be utilized such that smallerquantities of drug are added to blood.

Table 3 illustrates the utility of2-[4((((3,5-dimethylphenyl)amino)carbonyl)methyl)phenoxy]-2-methylpropionic acid (called RSR-13) in preserving the oxygen affinity ofhemoglobin in stored blood. RSR-13, 1 mM and 2 mM, was added to samplesof human RBCs (packed cells) which were stored at 4° C. in standardadsol formulation for 40-70 days. As can be seen from Table 3, the ODCof untreated blood left-shifts over time (indicated by a drop in the P₅₀value) to a high oxygen affinity state. The increase in oxygen affinityof stored blood is attributed to a decreased concentration of 2,3-DPG.The P₅₀ value of 40 day old untreated samples left shifted to 32;however, samples treated with 1 mM RSR-13 remained relatively unchanged(P₅₀ =90) and those treated with 2 mM RSR-13 were right shifted (P₅₀=45). Table 3 shows similar concentration dependent effects of RSR-13 onthe ODCs of 50, 60, 70 day old packed cells. Because of the glycolyticmetabolism, the pH of untreated red cells dropped over a period of timefrom 6.85 at 40 days to 6.6 for 70 day old samples and this wouldpossibly explain the slight right shifting of untreated 70 day oldsamples compared to 40 day old samples under the Bohr effect. The pH ofred blood cells treated with RSR-13 was consistently lower thanuntreated samples, which suggests that RSR-13 favorably decreases therate of glycolytic metabolism. RSR-13 had no adverse effect on thestability of RBCs as evidenced by consistent RBC counts in treated anduntreated samples. Similarly, the amount of hemolysis was consistent inboth treated and untreated samples of packed cells.

FIG. 11 shows the percentage oxygen delivered, ΔY, by packed cells.Changes in the oxygen saturation ΔY was calculated by Hill's equation(discussed in Stryer, Biochemistry, W. H. Freeman and Co., SanFrancisco, 1975, Chapter 4, pp, 71-94, which are herein incorporated byreference) at 100 to 30 torr. Column 1 shows the ΔY (59) correspondingto the untreated packed red blood cells. Column 2 shows the ΔY (50) ofpacked red blood cells stored for 40 days at 4° C. in the best availableadsol formulation. Column 3 shows that the ΔY=58 for 40 day old packedcells treated with RSR-13 (1 mM), which is comparable to fresh packedcells. Note, that the decrease (approximately 10%) in the oxygendelivery by packed cells is corrected by the addition of 1 mmol RSR-13.

Table 4 shows the change in the P₅₀ values of outdated packed red bloodcells on treatment with2-[4-((((3,5-dimethylphenyl)amino)carbonyl)methyl)phenoxy]-2-methylpropionic acid (RSR-13). 50 μl of 40, 50, and 60 day old red cells weremixed with 50 μl of RSR-13 to give final concentrations of RSR-13 at 1mmol and 2 mmol. Control samples were prepared by mixing 1:1 packedcells and buffer. As can be seen from FIG. 15, the P₃₀ value ofuntreated samples were consistently lower than samples treated withRSR-13. In addition, a comparison of the results for the fresh red cellswith red cells which were aged 40, 50, and 60 days shows a sharp declinein P₅₀ value with age. The P₅₀ values of 40, 50 , 60 day old red cellsamples treated with 1 mmol RSR-13 were comparable to the P₅₀ =38 valuefound for fresh red cells. These results show that the addition ofRSR-13 to the stored red cells restores the cells oxygen affinity.

Since the compounds contemplated by this invention are capable ofallosterically modifying hemoglobin so that a low oxygen affinity "T"state is favored (right shifting the equilibrium curve as indicated bythe P₅₀ column in Tables 1-2), these compounds will be useful intreating a variety of disease states in mammals including humans wheretissues suffer from low oxygen tension, such as cancer and ischemia. Aspointed out by Hirst et al. in Radiat. Res., Vol. 112, (1987), pp. 164,decreasing the oxygen affinity of hemoglobin in circulating blood hasbeen shown to be beneficial in the radiotherapy of tumors. The compoundsmay be administered to patients in whom the affinity of hemoglobin foroxygen is abnormally high. Particular conditions include certainhemoglobinopathies and certain respiratory distress syndromes in newborn infants aggravated by high fetal hemoglobin levels and when theavailability of hemoglobin/oxygen to the tissues is decreased (e.g., inischemic conditions such as peripheral vascular disease, coronaryocclusion, cerebral vascular accidents, or tissue transplant). Thecompounds may also be used to inhibit platelet aggregation and may beused for antithrombotic purposes and wound healing. Topical applicationcould be used for wound healing. In addition, the compounds may be usedto treat low oxygen related disorders in the brain such as Alzheimer'sdisease, depression, and schizophrenia. It may be desirable toadminister the compounds to a patient prior to and/or simultaneouslywith the transfusion of the treated whole blood or red blood cells inorder to avoid substantial variations in the hemoglobin oxygen affinitydue to dilution that occurs when the blood is administered.

The compounds can be added to whole blood or packed cells preferably atthe time of storage or at the time of transfusion in order to facilitatethe dissociation of oxygen from hemoglobin and improve the oxygendelivering capability of the blood. Preferably, the compounds would beadded in an amount of about 50 mg to 1 g per unit of blood (473 ml) orunit of packed cells (235 ml). When blood is stored, the hemoglobin inthe blood tends to increase its affinity for oxygen by losing2,3-diphosphoglycerides. As described above, the compounds of thisinvention are capable of reversing and/or preventing the functionalabnormality of hemoglobin which is observed when whole blood or packedcells are stored. The compounds may be added to whole blood or red bloodcell fractions in a closed system using an appropriate reservoir inwhich the compound is placed prior to storage or which is present in theanticoagulating solution in the blood collecting bag.

Administration can be achieved orally, by intravenous or intraperitonealinjection, or rectally by suppository where the dose and the dosingregiment is varied according to individual sensitivity and the type ofdisease state being treated. Studies with mice have shown that amg/kg/day dose of2-[4((((3,5-dimethylphenyl)amino)carbonyl)methyl)phenoxy]-2-methylpropionic acid (C₂₀ H₂₃ NO₄) (discussed in Example 15) givenintraperitoneally is well tolerated. If the compounds are used for woundhealing, the compounds could advantageously be applied topicallydirectly to the wound area. In addition, the compounds can be mixed withblood external to a patient's body prior to and/or simultaneously with atransfusion. The compounds can be administered in the pure form or in apharmaceutically acceptable formulation including suitable elixirs,binders, and the like or as pharmaceutically acceptable salts or otherderivatives. It should be understood that the pharmaceuticallyacceptable formulations and salts include liquid and solid materialsconventionally utilized to prepare injectable dosage forms and soliddosage forms such as tablets and capsules. Water may be used for thepreparation of injectable compositions which may also includeconventional buffers and agents to render the injectable compositionisotonic. Solid diluents and excipients include lactose starch,conventional disintegrating agents, coatings and the like.

While the invention has been described in terms of its preferredembodiments, those skilled in the art will recognize that the inventioncan be practiced with modification within the spirit and scope of theappended claims.

Having thus described our invention, what we claim as new and desire tosecure by Letters Patent is as follows:
 1. A compound of the generalstructural formula: ##STR5## wherein R₃₋₆ are either hydrogen, halogen,or a substituted or unsubstituted C₁₋₃ alkyl group, or a C₁₋₃ ether orester, and these moieties may be the same or different, or alkylmoieties of an aromatic or aliphatic ring incorporating two of the R₃₋₆sites, andwherein R₁ has the formula: ##STR6## where R₁ can be connectedto any position on the phenyl ring, and where R₇ and R₈ are hydrogen,halogen, methyl, or ethyl groups and these moieties may be the same ordifferent, or alkyl moieties as part of an aliphatic ring connecting R₇and R₈, and where R₉ is a hydrogen, halogen, C₁₋₃ loweralkyl, or a saltcation.